Project Summary: The objective of this proposal is to define the role of damaged RNA in mediating signaling events during cancer chemotherapy. In the previous funding cycle of this grant, we uncovered a novel ubiquitin-dependent signaling pathway initiated specifically when cells encounter alkylating agents, one of the most commonly used systemic modalities for cancer treatment. This pathway depends on the RNF113A E3 ubiquitin ligase, which in turn recruits the ASCC DNA helicase complex as well as the ALKBH3 demethylase/dealkylase. To date, most of the studies on alkylation damage, including our own work, has focused on how alkylated lesions are recognized and repaired on DNA. Yet, damage to RNA is far more abundant than damaged DNA in any cell which encounters an alkylating agent. Using novel tools to induce or repair alkylated RNA, we have found that alkylated RNA is necessary and sufficient to activate the E3 ligase function of RNF113A and recruit the ASCC complex. Our evidence also indicates that RNF113A is a phospho-activated E3 ligase, and that the CDK12 kinase is responsible for its phosphorylation. This data serves as the basis for our model that RNA alkylation functions upstream of a kinase (CDK12) that activates an E3 ligase (RNF113A), which in turn recruits an alkylation repair complex (ASCC-ALKBH3). In this proposal, we will seek to understand the mechanistic basis for how RNA alkylation recapitulates the activation of the RNF113A-ASCC-ALKBH3 pathway (Aim 1). We will characterize the role of this pathway in removing damaged RNAs, preventing replication- transcription conflicts and RNA-DNA hybrids (R-loops) (Aim 2). Finally, we will determine the functional consequences of CDK12-mediated phosphorylation in the activation of RNF113A and its role in tumor chemotherapy responses (Aim 3). Notably, since small molecule CDK12 inhibitors are already available, we will be able to test whether targeting this pathway will improve alkylation therapy responses. Together, our work will establish a new paradigm in nucleic acid damage signaling, where RNA damage plays a central role in mediating DNA repair.